Biology subjects

Heemstra, J. M.

Publications and source records attributed to Heemstra, J. M..

5 recordsLinked to original sources

bioRxiv2026

Chemical Lithography: Selective Glyoxal Caging of mRNAs to Control Gene Expression

Achieving predictable, tunable, and temporal control over mRNA function would grant direct regulation of gene expression, facilitating the development of new therapeutics and biotechnologies. Although several approaches for stimuli-responsive control of nucleic acids have been explored, most are limited to short oligonucleotides, lack a timed-release mechanism, or both. We envisioned a complementary method using glyoxal as a caging reagent. Glyoxal readily reacts with amidine groups found on the faces of nucleobases to give stable bis-hemiaminal adducts, directly disrupting hydrogen bonding. Fortuitously, this reaction is readily reversible, enabling spontaneous time-release decaging that varies with temperature. However, when applied previously to full-length mRNAs, the sequence length and excessive adduct formation resulted in no reactivation under relevant physiological conditions. To address this challenge, we developed chemical lithography in which portions of longer RNAs are "masked" through hybridization to complementary DNAs, permitting selective caging on only non-masked regions and preventing excessive adduct formation. We present an optimized glyoxalation protocol applied to EGFP as a model mRNA sequence and evaluate masking effectiveness through qualitative and quantitative studies. Using EGFP fluorescence, we monitored and assessed the ability of selective glyoxalation to control gene expression over time in vitro. We demonstrate the direct dependence of both the initial inhibited expression and the respective activity recovery based on the amount and location of glyoxalation. We also highlight distinct caging patterns exhibiting total inhibition upon initial treatment and complete reactivation following decaging. We anticipate that this approach will improve the mechanistic study of mRNA and gene expression and also facilitate new investigations and methods within chemical biology and biomedicine.

biochemistry↗

bioRxiv2026

dsRADAR: Imaging and Quantifying Cellular dsRNA by Repurposing RNA Binding Proteins

Double-stranded RNA (dsRNA) is recognized by cellular receptors as a sign of viral infection, triggering the innate immune response. Increasing evidence shows that cellular dysregulation, for example in immune disorders and neurodegenerative diseases, can also lead to accumulation of endogenously produced dsRNA that stimulates a viral-like immune response. Additionally, dsRNA contamination in RNA therapeutics can lead to harmful side effects via a similar pathway. Despite the clinical relevance of dsRNA, reliable tools for its detection remain limited. At present, dsRNA detection relies almost exclusively on the monoclonal antibodies J2 and K1, which suffer from sequence bias and low sensitivity, limiting their reliability. To address this challenge, we aimed to repurpose naturally occurring dsRNA-binding domains (dsRBDs) to produce reliable, pan-specific affinity reagents for dsRNA. We first systematically screened the dsRBDs of the three human adenosine deaminases acting on RNA (ADARs). This analysis identified ADAR3 dsRBDs as promising candidates due to their reduced sequence dependence compared to the dsRBDs of ADAR1 and ADAR2. We then engineered ADAR3-derived dsRBD constructs having varying linker lengths and domain combinations, allowing us to specifically vary the length cutoff of dsRNA detected, thus creating dsRNA accumulation detected by ADAR3 RBDs (dsRADAR) affinity reagents. Finally, we demonstrate the superior performance of dsRADAR over currently available dsRNA antibodies in a cell model of viral infection and a tissue model of gastric inflammation. Together, dsRADAR provides a sensitive and reliable approach for imaging and quantifying diverse dsRNA structures in a variety of biological contexts. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/724404v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d89c30org.highwire.dtl.DTLVardef@1f64fc1org.highwire.dtl.DTLVardef@1ee391forg.highwire.dtl.DTLVardef@e834a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Illuminating the Role of A-to-I Editing in Gastric Cancer using EndoVIA 2.0

Background & AimsAdenosine-to-inosine (A-to-I) RNA editing is an essential post-transcriptional modification catalyzed by ADAR enzymes, and emerging evidence suggests its dysregulation can contribute to cancer. However, technical barriers have hindered spatial analysis of editing activity in formalin-fixed paraffin-embedded (FFPE) tissues--an abundant but challenging sample type. Here, we introduce EndoVIA 2.0, an optimized immunostaining assay that enables spatial detection of edited RNAs in FFPE tissues. MethodsUsing human Endonuclease V (hEndoV) as a molecular affinity reagent, we developed a calcium-dependent staining workflow compatible with crosslinked tissues. EndoVIA 2.0 was validated in cell lines with altered ADAR1 expression, fresh frozen tissues, gastric organoids, and FFPE tissue sections. Quantitative imaging was combined with machine-learning segmentation and spatial analysis. ResultsEndoVIA 2.0 successfully detected A-to-I editing changes in ADAR1 knockout and overexpression models and revealed differential editing patterns across tissues. In gastric organoids and FFPE tissues, the assay spatially resolved editing heterogeneity and distinguished ADAR1-deficient from ADAR1-sufficient cells. Application to long-archived lung and breast cancer FFPE tissues suggest EndoVIA 2.0s broad utility and potential of capturing disease-associated hyper-editing in malignant samples. ConclusionsEndoVIA 2.0 enables robust, spatial detection of A-to-I editing in FFPE tissues--circumventing the limitations of RNA extraction and unlocking access to archived clinical specimens. This platform lays the foundation for mapping RNA editing dynamics in cancer progression and may support future biomarker discovery.

molecular biology↗

bioRxiv2024

Spatial visualization of A-to-I Editing in cells using Endonuclease V Immunostaining Assay (EndoVIA)

Adenosine-to-Inosine (A-to-I) editing is one of the most widespread post-transcriptional RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Varying across tissue types, A-to-I editing is essential for numerous biological functions and dysregulation leads to autoimmune and neurological disorders, as well as cancer. Recent evidence has also revealed a link between RNA localization and A-to-I editing, yet understanding of the mechanisms underlying this relationship and its biological impact remains limited. Current methods rely primarily on in vitro characterization of extracted RNA that ultimately erases subcellular localization and cell-to-cell heterogeneity. To address these challenges, we have repurposed Endonuclease V (EndoV), a magnesium dependent ribonuclease that cleaves inosine bases in edited RNA, to selectively bind and detect A-to-I edited RNA in cells. The work herein introduces Endonuclease V Immunostaining Assay (EndoVIA), a workflow that provides spatial visualization of edited transcripts, enables rapid quantification of overall inosine abundance, and maps the landscape of A-to-I editing within the transcriptome at the nanoscopic level.

molecular biology↗

bioRxiv2019

Selective enrichment of A-to-I edited transcripts from cellular RNA using Endonuclease V

Immunoprecipitation enrichment has significantly improved the sensitivity and accuracy of detecting RNA modifications in the transcriptome. However, there are no existing methods for selectively isolating adenosine-to-inosine (A-to-I) edited RNAs. Here we show that Escherichia coli Endonuclease V (eEndoV), an inosine-cleaving enzyme, can be repurposed to bind and isolate A-to-I edited transcripts from cellular RNA through adjustment of cationic conditions. While Mg2+ is required for eEndoV catalysis, it has also been shown that similar levels of Ca2+ instead promote binding of inosine without cleavage. Leveraging these properties, we observe that Ca2+-supplemented eEndoV is highly specific for inosine in RNA and exhibits low nanomolar binding affinity. We then demonstrate EndoVIPER (Endonuclease V inosine precipitation enrichment) as a facile and robust method to isolate A-to-I edited transcripts from cellular RNA. We envision the use of this approach as a straightforward and cost-effective strategy to enrich edited RNAs and detect A-to-I sites with improved sensitivity and fidelity.

molecular biology↗